Delivery of human factor IX in mice by encapsulated recombinant myoblasts: a novel approach towards allogeneic gene therapy of hemophilia B

A potentially cost-effective strategy for gene therapy of hemophilia B is to create universal factor IX-secreting cell lines suitable for implantation into different patients. To avoid graft rejection, the implanted cells are enclosed in alginate-polylysine-alginate microcapsules that are permeable to factor IX diffusion, but impermeable to the hosts' immune mediators. This nonautologous approach was assessed by implanting encapsulated mouse myoblasts secreting human factor IX into allogeneic mice. Human factor IX was detected in the mouse plasma for up to 14 days maximally at approximately 4 ng/mL. Antibodies to human factor IX were detected after 3 weeks at escalating levels, which were sustained throughout the entire experiment (213 days). The antibodies accelerated the clearance of human factor IX from the circulation of the implanted mice and inhibited the detection of human factor IX in the mice plasma in vitro. The encapsulated myoblasts retrieved periodically from the implanted mice up to 213 days postimplantation were viable and continued to secrete human factor IX ex vivo at undiminished rates, hence suggesting continued factor IX gene expression in vivo. Thus, this allogeneic gene therapy strategy represents a potentially feasible alternative to autologous approaches for the treatment of hemophilia B.

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Mesenchymal Stromal Cells Gene-Enhanced To Express Human Factor IX and Their In Vivo Use for Correction of Hemophilia B Phenotype in R333Q Mice.

Blood ◽

10.1182/blood.v110.11.5136.5136 ◽

2007 ◽

Vol 110 (11) ◽

pp. 5136-5136

Author(s):

Daniel L. Coutu ◽

Jessica Cuerquis ◽

May Griffith ◽

Mark D. Blostein ◽

Jacques Galipeau

Keyword(s):

Gene Therapy ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Human Factor ◽

Factor Ix ◽

Hemophilia B ◽

Monogenic Disease ◽

Human Factor Ix ◽

B Mice

Abstract Hemophilia B is considered an appropriate disease target for gene therapy because it is a well characterized monogenic disease with a large therapeutic index. Despite promising preclinical and clinical trials in the last decade, safety and efficacy concerns associated with the in vivo administration of viral vectors still need to be addressed before gene therapy becomes part of the standard arsenal for clinicians. Our laboratory has developed a cell therapy approach using gene-enhanced autologous Mesenchymal Stromal Cells (MSCs) to deliver a therapeutic plasmatic protein which addresses these safety concerns. In this study, we tested whether MSCs engineered to express human Factor IX (hFIX) can be used to reverse the bleeding phenotype of R333Q hemophilia B mice developed by Stafford et al. We retrovirally engineered MSCs harvested from normal C57Bl/6 to express hFIX. A gene enhanced polyclonal population of MSCs was capable of producing carboxylated and fully active hFIX by in vitro clotting assays. By ELISA, the cells were shown to produce approximately 250ng of hFIX per million cells per 24h. Ten million of these cells were embedded in a collagen I gel matrix and implanted subcutaneously in R333Q hemophilia B mice (n=10). hFIX activity in mouse plasma (test and control groups) were followed weekly by aPTT assays. hFIX activity reached levels as high as 20% normal activity in some animals with an average +/− SEM of 11.2 +/− 2.1 (FIX activity in controls is <1%). The hFIX activity returned to baseline within 4 weeks. In conclusion, we demonstrate that gene-enhanced autologous MSCs can serve as an effective delivery of functional FIX for temporary correction of the hemophilia B phenotype. We hypothesize the presence of GFP co-expression by the implanted MSCs caused their immune rejection and we are currently testing this hypothesis.

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Pharmacokinetics of Human Factor IX in a dog with Severe Hemophilia B.

10.1055/s-0039-1682760 ◽

1977 ◽

Author(s):

P.A. Gentry ◽

A.R. Thompson ◽

A.W. Forrey

Keyword(s):

Human Factor ◽

Numerical Procedure ◽

Factor Ix ◽

Hemophilia B ◽

High Yield ◽

Severe Hemophilia ◽

Activity Data ◽

Human Factor Ix

In preparing a factor IX concentrate with a high yield and low hepatitis and thromboembolic risks, we have tested this material for survival in an in vivo system, the hemophiliac dog. By following the disappearance of radiolabeled, isolated factor IX in addition to the classic clotting assays, data on protein survival and more accurate kinetic parameters were obtained.Crude factor IX concentrate was prepared by batchwise adsorption-elution with DEAE-Sephadex using cryoprecipitate-poor human plasma. Isolated human factor IX was radiolabeled with 125I by chloramine-T without in vitro loss of clotting activity (Thompson, J Clin Invest, in press, 1977). A preparation containing both crude and isolated factor IX was then subjected to filtration (0.22 μm) and lyophilization; clotting and radioactivity were not altered by these steps.Following infusion of the combined preparation into a dog with severe hemophilia B (0% baseline factor IX) 10 post infusion samples were taken over 96 h for determination of radioactivity and factor IX clotting activity. These data were then analyzed by fitting to a two exponential expression using a Marquart non-linear least squares numerical procedure for a two compartment open model. The central volume was 14.5% of the animal’s body weight; the total volume of distribution was 28% with a t 1/2 distribution of 114 min. The t 1/2 elimination was 20 h; the slower phase of elimination (β, or that affected by redistribution) had a t 1/2 of 40 h. Factor IX clotting activity from the crude concentrate closely paralleled radioactivity from the isolated factor IX throughout the 96 h; t 1/2 β was slightly longer from the clotting activity data.

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Production of human factor IX in animals by genetically modified skin fibroblasts: potential therapy for hemophilia B

Blood ◽

10.1182/blood.v73.2.438.bloodjournal732438 ◽

1989 ◽

Vol 73 (2) ◽

pp. 438-445

Author(s):

TD Palmer ◽

AR Thompson ◽

AD Miller

Keyword(s):

Human Factor ◽

Normal Skin ◽

Human Fibroblasts ◽

Retroviral Vectors ◽

Factor Ix ◽

Skin Fibroblasts ◽

Hemophilia B ◽

Clotting Factor ◽

Human Factor Ix

Inherited diseases might be treated by introducing normal genes into a patient's somatic tissues to correct the genetic defects. In the case of hemophilia resulting from a missing clotting factor, the required gene could be introduced into any cell as long as active factor reached the circulation. We previously showed that retroviral vectors can efficiently transfer genes into normal skin fibroblasts and that the infected cells can produce high levels of a therapeutic product in vitro. In the current study, we examined the ability of skin fibroblasts to secrete active clotting factor after infection with different retroviral vectors encoding human clotting factor IX. Normal human fibroblasts infected with one vector secreted greater than 3 micrograms factor IX/10(6) cells/24 h. Of this protein, greater than 70% was structurally and functionally indistinguishable from human factor IX derived from normal plasma. This suggests that infected autologous fibroblasts might provide therapeutic levels of factor IX if transplanted into patients suffering from hemophilia B. By transplanting normal diploid fibroblasts infected with the factor IX vectors, we showed that human factor IX can be produced and is circulated at readily detectable levels in rats and mice.

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Adenovirus-mediated regulatable Expression of human Factor IX in vitro and in vivo

32nd Hemophilia Symposium Hamburg 2001 ◽

10.1007/978-3-642-18150-4_9 ◽

2003 ◽

pp. 72-80

Author(s):

M. A. Srour ◽

H. Fechner ◽

X. Wang ◽

U. Siemetzki ◽

T. Albert ◽

...

Keyword(s):

Human Factor ◽

Factor Ix ◽

Human Factor Ix

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210. Neonatal Gene Therapy with a Human Factor IX-Expressing Retroviral Vector Prevents Antibody Responses in Dogs and Hemophilia B Mice

Molecular Therapy ◽

10.1016/s1525-0016(16)40652-0 ◽

2003 ◽

Vol 7 (5) ◽

pp. S83

Keyword(s):

Gene Therapy ◽

Human Factor ◽

Retroviral Vector ◽

Factor Ix ◽

Antibody Responses ◽

Hemophilia B ◽

Human Factor Ix ◽

B Mice

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Production of human factor IX in animals by genetically modified skin fibroblasts: potential therapy for hemophilia B

Blood ◽

10.1182/blood.v73.2.438.438 ◽

1989 ◽

Vol 73 (2) ◽

pp. 438-445 ◽

Cited By ~ 121

Author(s):

TD Palmer ◽

AR Thompson ◽

AD Miller

Keyword(s):

Human Factor ◽

Normal Skin ◽

Human Fibroblasts ◽

Retroviral Vectors ◽

Factor Ix ◽

Skin Fibroblasts ◽

Hemophilia B ◽

Clotting Factor ◽

Human Factor Ix

Abstract Inherited diseases might be treated by introducing normal genes into a patient's somatic tissues to correct the genetic defects. In the case of hemophilia resulting from a missing clotting factor, the required gene could be introduced into any cell as long as active factor reached the circulation. We previously showed that retroviral vectors can efficiently transfer genes into normal skin fibroblasts and that the infected cells can produce high levels of a therapeutic product in vitro. In the current study, we examined the ability of skin fibroblasts to secrete active clotting factor after infection with different retroviral vectors encoding human clotting factor IX. Normal human fibroblasts infected with one vector secreted greater than 3 micrograms factor IX/10(6) cells/24 h. Of this protein, greater than 70% was structurally and functionally indistinguishable from human factor IX derived from normal plasma. This suggests that infected autologous fibroblasts might provide therapeutic levels of factor IX if transplanted into patients suffering from hemophilia B. By transplanting normal diploid fibroblasts infected with the factor IX vectors, we showed that human factor IX can be produced and is circulated at readily detectable levels in rats and mice.

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Neonatal or hepatocyte growth factor–potentiated adult gene therapy with a retroviral vector results in therapeutic levels of canine factor IX for hemophilia B

Blood ◽

10.1182/blood-2002-10-3050 ◽

2003 ◽

Vol 101 (10) ◽

pp. 3924-3932 ◽

Cited By ~ 73

Author(s):

Lingfei Xu ◽

Cuihua Gao ◽

Mark S. Sands ◽

Shi-Rong Cai ◽

Timothy C. Nichols ◽

...

Keyword(s):

Gene Therapy ◽

Growth Factor ◽

Gene Transfer ◽

Hepatocyte Growth Factor ◽

Retroviral Vector ◽

Factor Ix ◽

Hemophilia B ◽

Hepatocyte Growth

AbstractHemophilia B is a bleeding disorder resulting from factor IX (FIX) deficiency that might be treated with gene therapy. Neonatal delivery would correct the disease sooner than would transfer into adults, and could reduce immunological responses. Neonatal mice were injected intravenously with a Moloney murine leukemia virus–based retroviral vector (RV) expressing canine FIX (cFIX). They achieved 150% to 280% of normal cFIX antigen levels in plasma (100% is 5 μg/mL), which was functional in vitro and in vivo. Three newborn hemophilia B dogs that were injected intravenously with RV achieved 12% to 36% of normal cFIX antigen levels, which improved coagulation tests. Only one mild bleed has occurred during 14 total months of evaluation. This is the first demonstration of prolonged expression after neonatal gene therapy for hemophilia B in mice or dogs. Most animals failed to make antibodies to cFIX, demonstrating that neonatal gene transfer may induce tolerance. Although hepatocytes from newborns replicate, those from adults do not. Adult mice therefore received hepatocyte growth factor to induce hepatocyte replication prior to intravenous injection of RV. This resulted in expression of 35% of normal cFIX antigen levels for 11 months, although all mice produced anti-cFIX antibodies. This is the first demonstration that high levels of FIX activity can be achieved with an RV in adults without a partial hepatectomy to induce hepatocyte replication. We conclude that RV-mediated hepatic gene therapy is effective for treating hemophilia B in mice and dogs, although the immune system may complicate gene transfer in adults.

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Self-complementary adeno-associated virus vectors containing a novel liver-specific human factor IX expression cassette enable highly efficient transduction of murine and nonhuman primate liver

Blood ◽

10.1182/blood-2005-10-4035 ◽

2006 ◽

Vol 107 (7) ◽

pp. 2653-2661 ◽

Cited By ~ 269

Author(s):

Amit C. Nathwani ◽

John T. Gray ◽

Catherine Y. C. Ng ◽

Junfang Zhou ◽

Yunyu Spence ◽

...

Keyword(s):

Gene Therapy ◽

Human Factor ◽

Factor Ix ◽

Expression Cassette ◽

Hemophilia B ◽

Adeno Associated Virus ◽

Knock Out ◽

Human Factor Ix ◽

Murine Liver ◽

Knock Out Mice

AbstractTransduction with recombinant adeno-associated virus (AAV) vectors is limited by the need to convert its single-stranded (ss) genome to transcriptionally active double-stranded (ds) forms. For AAV-mediated hemophilia B (HB) gene therapy, we have overcome this obstacle by constructing a liver-restricted mini–human factor IX (hFIX) expression cassette that can be packaged as complementary dimers within individual AAV particles. Molecular analysis of murine liver transduced with these self-complementary (sc) vectors demonstrated rapid formation of active ds-linear genomes that persisted stably as concatamers or monomeric circles. This unique property resulted in a 20-fold improvement in hFIX expression in mice over comparable ssAAV vectors. Administration of only 1 × 1010 scAAV particles led to expression of hFIX at supraphysiologic levels (8I U/mL) and correction of the bleeding diathesis in FIX knock-out mice. Of importance, therapeutic levels of hFIX (3%-30% of normal) were achieved in nonhuman primates using a significantly lower dose of scAAV than required with ssAAV. Furthermore, AAV5-pseudotyped scAAV vectors mediated successful transduction in macaques with pre-existing immunity to AAV8. Hence, this novel vector represents an important advance for hemophilia B gene therapy.

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